Proteins with mutations to decrease N-terminal methylation

ABSTRACT

This invention relates to proteins with N-terminal modifications in their amino acid sequence that reduce or eliminate the methylation of the N-terminal methionine that occurs when such proteins are produced in bacteria such as E. coli.

This invention relates to proteins with N-terminal modifications intheir amino acid sequence that decrease or eliminate the methylation ofthe N-terminal amino acid that occurs when such proteins are produced inbacteria such as E. coli.

BACKGROUND

Methylation of the N-terminal amino acid for many proteins has beenobserved, including when the N-terminal amino acid is methionine,alanine, phenylalanine and proline (Stock et al., (1987) FEBS Letters220:8-14). In pilins expressed in Pseudomonas aeruginosa, themethylation of a number of different N-terminal amino acids has beenpostulated to be dependent on a fifth position glutamate (Pasloske andParanchych, (1988) Molecular Microbiol. 2(4):489-495). Amino acids thatwere methylated at the N-terminus were alanine, glycine, tyrosine andmethionine, while serine and phenylalanine were not (Strom and Lory,(1991) J. Biol. Chem. 266:1656-1664).

Recently, several signal transduction proteins have been shown to bemethylated at glutamic acid side chains and/or at C-terminal amino acids(Stock and Lukat, (1991) Ann. Rev. Biophys. Chem. 20:109-136; Morgan etal., (1993) J. Bacteriol. 175:133-140). In bacteria, the activities ofsignal transducing proteins are regulated by methylesterification atglutamic acid side chains. Methyl accepting chemotaxis proteins areimmunogenic and demonstrate high antigenic relatedness (Morgan et al.,(1993) J. Bacteriol. 175:133-140). Antibodies against one methylatedprotein can crossreact with a very distinct spectrum of other methylatedproteins with sequence identities of only 60 percent (Morgan et al.,(1993) J. Bacteriol. 175:133-140). Paik, (1984) Methods Enzymol.106:265-268, has suggested that methylation of the N-terminal amino acidmay affect the physiological characteristics of proteins.

Recently, N-monomethylmethionine was observed at the N-termini of theribosomal protein L16 (Brosius and Chen, (1976) FEBS Letters 62:105-109)and the bacterial chemotaxis protein CheZ (Stock et al., (1987) J. Biol.Chem. 262:8011-8014; Stock and Stock, (1987) J. Bacteriol.169:3301-3311). There is some sequence similarity between these twoproteins at the N-terminal end:

    L16 (E. coli) N-methyl-Met-Leu-Gln-Pro-                    (SEQ. ID NO. 1)

    CheZ (E. coli) N-methyl-Met-Met-Gln-Pro-                   (SEQ. ID NO. 2)

Stack, (1987) Advances in Post-translational Modification of Proteinsand Aging, edited by Zappia, pp. 387-399, has suggested that theN-terminal sequence in CheZ and L16 is a signal of methylation forN-terminal methionine. The level of methylation reported in theliterature is in the range of 30-50 percent. The mechanism ofmethylation is unknown, although it is known that it is dependent onS-adenosylmethionine. Because of CheZ, the process of methylation waslinked with chemotaxis, but experiments indicated that no chemotaxisproteins are involved in methylation of CheZ. CheZ was expressed from aplasmid vector with the CheZ gene inserted behind the lac promoter in E.coli (JM109) that produces no flagellar or chemotaxis proteins. Thefirst cycle of Edman degradation of this protein produced equal amountsof pth-Met and pth- N-methylmethionine (Stock et al., (1987) J. Biol.Chem. 262:8011-8014). Thus, CheZ methylation does not appear to becatalyzed by any other chemotaxis protein. It has been suggested thatCheZ is self methylated or there is another unidentifiedmethyl-transferase different than glutamate methyl-transferase involvedin methylation of methionine. In addition, IF-3 is an E. coli proteinthat presents an N-terminal methylmethionine, but it has a glycine inposition 4 (Brauer et al., (1977) FEBS Lett. 79:269-275).

There are at least ten other sequences of L16 which have been reportedrecently, but unfortunately all of them are translations of nucleic acidsequences (Atlas of Proteins and Genomic Sequences (1992) NationalResearch Foundation, Compact Disc Edition). In most of them, Gln in theposition 3 is mutated to Ser, which makes the sequences identical withthe sequence of the dialpha chain of a recombinant hemoglobin known asrHb1.1 (Hoffman et al., WO 90/13645, published Nov. 15, 1990).

    L16 sequences Met-Leu-Ser-Pro-                             (SEQ. ID NO. 3)

    rHb1.1 (dialpha chain) Met-Leu-Ser-Pro-                    (SEQ. ID NO. 4)

Because these protein sequences are translations of gene sequences,there has not been any information about methionine methylation untilnow. DiDonato et al., (1983) J. Biol. Chem. 258:11890-11895, haveachieved carboxymethylation of alpha amino groups of hemogobin usingchemical means (reductive alkylation). Brunner et al., achievedacetylation of heterologous peptides by using a growth media low inamino acids (PCT Application WO 90/10706, published Sep. 20, 1990).

Besides the extent of methylation of an N-terminal amino acid, theextent to which the N-teminal methionine is processed (removed) has beenpostulated to be affected by the amino acid in position 2 (Hirel, etal., (1989) Proc. Natl. Acad. Sci USA 86:8247-8251). It was shown thatglycine, alanine, proline, serine, threonine and cysteine appear toinitiate N-terminal methionine processing, while Fujiyama and Tamanoi,(1990) J. Biol. Chem. 265:3362-3368 showed that RAS2, a S. cerevisiaeprotein undergoes N-terminal methionine removal with a proline inposition 2.

SUMMARY OF THE INVENTION

The present invention includes a method for decreasing methylation of anN-terminal methionine of a protein comprising: mutating a proline atamino acid position 4 of said protein to a non-proline residue, whereinsaid protein is at least partially methylated at the N-terminalmethionine prior to said alteration, when said protein is expressed in abacterium.

A preferred aspect of the present invention is method for decreasingmethylation of an N-terminal methionine of a protein comprising:mutating a proline at amino acid position 4 of said protein to anon-proline residue, wherein said protein is at least partiallymethylated at the N-terminal methionine prior to said alteration, whensaid protein is expressed in a bacterium, wherein the mutating is a sitedirected mutagenesis protocol selected from the group consisting of theAmersham technique, the Promega technique, the PCR based site directedmutagenesis, and DNA casette mutagenesis and wherein the site directedmutagenesis of a proline at amino acid position 4 of said protein to anon-proline residue is selected from the group consisting of:

(a) substitution of the proline at amino acid position 4 with alanine,

(b) substitution of the proline at amino acid position 4 with serine,

(c) addition of alanine at amino acid position 2,

(d) addition of serine at amino acid position 2,

(e) addition of threonine at amino acid position 2, and

(f) addition of valine at amino acid position 2,

wherein said protein is at least 10 percent methylated at the N-terminalmethionine prior to said alteration, when said protein is expressed inE. coli.

Another aspect of the present invention is a demethylated proteincomprising a protein with decreased methylation obtained according tothe method aspect of the present invention, wherein said protein isselected from the group consisting of hemoglobin, L16 and CheZ.

A preferred aspect of this invention is a demethylated proteincomprising a protein with decreased methylation obtained according tothe method aspect of the present invention wherein said protein isrecombinant hemoglobin produced in E. coli.

Another aspect of the present invention is a pharmaceutical compositioncomprising a pharmaceutically effective amount of a demethylatedobtained according to the method aspect of the present invention.

To assist in the interpretation of the present patent, the followingterms shall have the following meaning throughout this patent, includingthe claims appended hereto, unless otherwise indicated.

"Hemoglobin" or "hemoglobin-like protein" comprises one or moreheterotetramers composed of (a) two alpha globin-like and two betaglobin-like polypeptides, (b) one di-alpha globin-like and two betaglobin-like polypeptides, (c) two alpha globin-like and one di-betaglobin-like polypeptide, (d) one di-alpha globin-like and one di-betaglobin-like polypeptides, (e) one fused alpha/beta globin-likepolypeptide and separate alpha and beta globin-like polypeptides, or (f)two fused alpha/beta globin-like polypeptides. A polypeptide of onetetramer may be crosslinked or genetically fused to a polypeptide ofanother tetramer. A hemoglobin is said to be multimeric if it comprisesmore than four globin subunits or domains. The term "multimeric" therebyincludes octameric hemoglobin (2 linked tetramers), as well as highermultimers. In hemoglobin or hemoglobin-like protein, whether derivedfrom natural or recombinant sources, in either the R or the T state,each alpha and beta globin-like polypeptide may contain a heme orprotoporphyrin IX prosthetic group and therefore may have the ability tobind oxygen.

"Recombinant hemoglobin" means hemoglobin comprising alpha and betaglobins at least one of which is obtained by expression of a globin genecarried by a recombinant DNA molecule, whether the hemoglobin is aconventional hemoglobin or a mutant species, resulting in expression ofa hemoglobin gene to produce a hemoglobin protein in a cell other than acell in which such hemoglobin gene and/or hemoglobin protein isnaturally found, i.e., the hemoglobin gene is heterologous to the hostin which it is expressed. Therefore, the expression of any humanhemoglobin gene in any cell other than a human red blood cell would beconsidered to be a recombinant hemoglobin. Moreover, the expression of avertebrate hemoglobin in any species of invertebrate, or any vertebrateother than the vertebrate where the hemoglobin to be expressed isnaturally occurring, would be considered a recombinant hemoglobin.Additionally, the expression of any naturally occurring hemoglobinmutant in any species other than the species in which it is naturallyoccurring, would be considered a recombinant hemoglobin. The expressionof any non-naturally occurring mutant hemoglobin in any species would beconsidered a recombinant hemoglobin.

"Genetically fused hemoglobin" means a hemoglobin-like proteincomprising at least one "genetically fused globin-like polypeptide"(globin pseudooligomer), the latter comprising two or more globin-likedomains which may be the same or different. A di-alpha globin-likepolypeptide is one which consists essentially of two alpha-globin-likepolypeptide sequences (domains) connected by peptide bonds between theC-terminus of the first alpha-globin-like polypeptide (domain) and theN-terminus of the second alpha-globin-like polypeptide (domain). Thesetwo sequences may be directly connected, or connected through a peptidelinker of one or more amino acids; the term "peptide bonds" is intendedto embrace both possibilities. Alpha globin chains crosslinked at the N-and C-termini other than by peptide bonds (e.g., by4,4'-diisothiocyanatostilbene-2,2'-disulfonates, DIDS) are not di-alphaglobins. The di-alpha globin-like polypeptide preferably is capable offolding together with beta globin and incorporating heme to formfunctional hemoglobin-like protein. The di-beta globin-like polypeptideis analogously defined. A di-alpha or di-beta globin-like polypeptidewith a mutation in only one of the component domains is called"asymmetric".

DESCRIPTION OF THE FIGURES

FIG. 1 shows a plasmid map of pSGE705, a plasmid used in the recombinantexpression of a mutant hemoglobin, rHb1.1. The plasmid map includesrelevant restriction sites.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to proteins with N-terminal modifications intheir amino acid sequence that reduce or eliminate the methylation ofthe N-terminal amino acid that occurs when such proteins are produced inbacteria such as E. coli. The N-terminal modifications comprise changingwhat we have discovered to be a critical portion of a methylation signal(namely the presence of a proline at amino acid position 4) for proteinsthat are produced in bacteria, preferably E. coli. Therefore, thepresent invention includes a method for decreasing methylation of anN-terminal methionine of a protein comprising: mutating a proline atamino acid position 4 of said protein to a non-proline residue, whereinsaid protein is at least partially methylated at the N-terminalmethionine prior to said alteration, when said protein is expressed in abacterium. Consequently, a protein that is partially methylated will,after being exposed to the method aspect of this invention, exist as ademethylated protein.

A partially methylated protein is a protein that has methylation of itsN-terminal amino acid, prefereably when the N-terminal amino acid is amethionine. Such a partially methylated protein is preferably methylatedas a result of a methylation enzyme, particularly when such methylationmechanism is regulated by a methylation signal in the amino acidsequence of the protein to be methylated. As has been discovered by theinventors of the present invention, such a methylation signal preferablycontains as its critical element a proline at amino acid position 4.That the present invention, in its method aspect, requires aqs astarting material a protein that is partially methylated means that theprotein need not be completely methylated. This is because whenmethylation is the result of a signal induced enzymatic methylation,there is often a contrary demethylating mechanism. Consequentlyequilibrium of the methylating and demethylating mechanism will resultin less than all of the protein being methylated. For purposes of thisinvention, a partially methylated protein is a protein in which theN-terminal methionine has at least 10 percent methylation, preferably atleast 20 percent methylation, more preferably at least 30 percentmethylation, most preferably about 35 percent methylation.

The method aspect of the present invention results in decreasedmethylation of a protein that is initially partially methylated.Consequently, decreasing methylation for a protein means a level ofmethylation that is less after the protein has been exposed to themethod of the present invention, preferably the level of methylation issignificantly less after the protein has been exposed to the method ofthe present invention. Significantly less methylation usually means thatthe amount of decrease in methylation is greater than the margin oferror for a particular measurement technique. Preferably the decrease inmethylation is to less than about 50 percent demethylation, morepreferably less than about 35 percent methylation, more preferably lessthan about 30 percent methylation, more preferably less than about 20percent methylation, more preferably less than about 10 percentmethylation, most preferably to a level of essentially nondetectablemethylation.

As discovered by the inventors of the present invention, the N-terminalmethionine of a dialpha chain of recombinant hemoglobin (rHb1.1; Hoffmanet al., WO 90/13645, published Nov. 15, 1990; Looker et al., (1992)Nature 356:258-260) is methylated with about 35 percent frequency whenproduced in E. coli, while the beta chain of rHb1.1 is free ofmethylation of the N-terminal methionine. This indicates that thesequences of the dialpha chain represent a site that is recognized by amethylation enzyme. The beta chain is similar in amino acid sequence,but it has the insertion of His so that Pro is moved into position 5from the N-terminal end.

    rHb1.1 (dialpha chain) CH.sub.3 -Met-Leu-Ser-Pro-          (SEQ. ID. NO. 5)

    rHb1.1 (beta chain) Met-His-Leu-Thr-Pro-                   (SEQ. ID. NO. 6)

This shifting of the position of the Pro destroys the methylation signaland the beta chain is not methylated. That the Leu in position 2 is notthe critical methylation signal is corraborated by the fact that CheZ ismethylated when produced in E. coli even though it has the N-terminalsequence of N-methyl-Met-Met-Gln-Pro-. Therefore, it appears that thePro in position 4 is critical to the methylation signal. Subsequentexperiments conducted by us (Example 8) have verified the criticality ofthe proline at position 4. There is no known naturally occurring humanhemoglobin with a mutation which either substitutes, deletes or shiftsthe Pro at position 4. A search of sequence data from other speciesindicates that there are five other species, which have the most similarsequences to human hemoglobin, and have a mutation at this position.Horse, donkey, kulan, zebra, gundi:

    Val-Leu-Ser-Ala-Ala-Asp-Lys-Thr-Asn-Val-Lys-Ala-Ala-Trp-   (SEQ. ID. NO. 7)

Apparently, alanine is the conserved substitution for proline inposition 4.

On the other hand, we have found that an antibody against E. coliproteins (ECP) can crossreact with the dialpha chain of rHb1.1 expressedin E. coli. This antibody does not crossreact with the dialpha chainexpressed in yeast or human hemoglobin alpha chain. Therefore, it ispossible that monocytes recognize the N-methylated terminal ofrecombinant rHb1.1 as E. coli protein and an immunogenic response isinduced.

Consequently, we have surprisingly found that the dialpha chain of arecombinant hemoglobin molecule rHb1.1 has a signal in its amino acidsequence that imparts a tendency for the N-terminal amino acid(methionine) to be methylated when the recombinant hemoglobin isproduced in bacteria. The tendency for methylation occurs at less than100 percent frequency and could not have been predicted at all from theteachings of the prior art. Therefore, the present invention not onlyteaches the occurrence of the N-terminal methylation in certainrecombinant hemoglobins, but it also teaches methods for preventing ordecreasing the N-methylation from recombinant hemoglobin molecules thatwould otherwise have such N-terminal methylation.

Consequently, the methods of the present invention can decrease themethylation of a protein, preferably a protein such as CheZ, L16 orhemoglobin, by mutating the proline at position 4 to a non-proline aminoacid to result in a demthylated protein. The demthylated proteinsolution can then be subjected to further purifications techniques thatare known in the art to further contaminants. When the protein ishemoglobin, it can be further purified to remove other hemoglobin andnon-hemoglobin contaminants from the demethylated hemoglobin solution toresult in a hemoglobin solution that is very pure. The techniques forfurther purification can be, for example, as described in Estep, U.S.Pat. Nos. 4,861,867 and 4,8331,012; Rausch et al., U.S. Pat. No.5,084,558 (human and mammalian sources), De Angelo et al., WO 93/08831and WO 91/16349; Hoffman et al., WO 90/13645 (yeast sources), Logan, etal., WO 92/22646 (transgenic systems) and Hoffman et al., WO 90/13645and Chivers and Belval, U.S. Ser. No. 08/097,273, filed Jul. 23, 1993(bacterial systems).

For the purposes of the present invention, functional hemoglobinsolution is any hemoglobin (desired or functional hemoglobin) that hasthe functionality necessary for a given utility. Utility of a purifiedhemoglobin solution can be, but is not limited to, reagent gradehemoglobin as a source of bio-available iron in dietary supplementation,as a highly purified molecular weight marker for laboratoryapplications, and most preferably as a modifier of the oxygen content ofa solution, such as in the case of use of hemoglobin as an oxygencarrying solution that enhances the oxygen content of blood. The desiredhemoglobin to be subjected to the demethylation method of the presentinvention can be either naturally occurring human hemoglobin or any of avariety of hemoglobin variants that are from other species, mutanthemoglobins, or hemoglobin-like molecules. The desired hemoglobin can beused alone in solution or can be part of a suitable pharmaceuticalcomposition such as those described in Hoffman and Nagai, U.S. Pat. No.5,028,588 and Chivers and Belval, U.S. Ser. No. 08/097,273, filed Jul.23, 1993.

The structure of conventional hemoglobin is well known. We herewithincorporate by reference the entire text of Bunn and Forget, eds.Hemoglobin: Molecular, Genetic and Clinical Aspects (W. B. Saunders Co.,Philadelphia, Pa.: 1986) and of Fermi and Perutz "Hemoglobin andMyoglobin," in Phillips and Richards, Atlas of Molecular Structures inBiology (Clarendon Press: 1981).

About 92% of the normal adult human hemolysate is Hb A (designatedalpha2 beta2, because it comprises two alpha and two beta chains). Thealpha chain consists of 141 amino acids. The iron atom of the heme(ferroprotoporphyrin IX) group is bound covalently to the imidazole ofhis 87 (the "proximal histidine"). The beta chain is 146 residues longand heme is bound to it at his 92.

The primary structure of a polypeptide is defined by its amino acidsequence and by identification of any modification of the side chains ofthe individual amino acids. The local bending of the chain is itssecondary structure. The tertiary structure of the hemoglobin moleculerefers to the steric relationships of amino acid residues, whilequaternary structure refers to the way in which the subunits (chains)are packed together. The tertiary and quaternary structure of thehemoglobin molecule have been discerned by X-ray diffraction analysis ofhemoglobin crystals, which allows one to calculate the three-dimensionalpositions of the atoms of the molecule.

Normal hemoglobin in vivo is retained within erythrocytes, which have alife span of about 180 days. When erythrocytes age and die, they releasehemoglobin into the bloodstream. There it dissociates into alpha-betadimers. The dimers are cleared either by renal filtration, or as aresult of haptoglobin binding. Hemoglobin may also be removed from serumby other mechanisms, such as by liver parenchymal cell uptake of freehemoglobin. The term "hemoglobin" as used in this application refers toa family of related molecules.

An alpha globin-like domain or polypeptide is a native alpha globin or amutant thereof differing from the native sequence by one or moresubstitutions, deletions or insertions, while remaining substantiallyhomologous (as hereafter defined) with human alpha globin, and stillcapable of associating with beta globin. A beta globin-like domain orpolypeptide is analogously defined. Subunits of animal hemoglobins ormutants thereof which are sufficiently homologous with alpha or betaglobin are embraced by the term "human alpha or beta globin-like domainor polypeptide." For example, the subunits of bovine hemoglobin arewithin the scope of these terms. The alpha- and beta-globin-likepolypeptides may be referred to collectively as "globins". For the sakeof convenience the term "polypeptide" may refer to a unitary chain or toa domain of a longer polypeptide chain. Preferably, the globin-likedomain or polypeptide has the ability to incorporate heme.

It is also possible to provide an "alpha/beta-globin-like pseudodimer"in which an alpha globin-like sequence is connected by peptide bonds toa beta globin-like sequence. This "alpha/beta globin-like polypeptide",and the di-alpha and di-beta globin-like polypeptides, may collectivelybe referred to as "pseudodimeric globin-like polypeptides" or as"diglobins". By extension, a hemoglobin-like protein comprising adi-alpha, a di-beta, or a alpha/beta globin-like polypeptide is a"pseudotetramer".

Even though the di-alpha hemoglobin does not dissociate into dimers, itis still cleared from the bloodstream, albeit more slowly than is thecase for normal hemoglobin.

In determining whether a polypeptide is substantially homologous toalpha (or beta) globin, sequence similarity is an important but notexclusive criterion. Sequence similarity may be determined byconventional algorithms, which typically allow introduction of a smallnumber of gaps in order to achieve the best fit. Preferably, thealpha-globin-like polypeptides (or domains thereof) of the presentinvention have at least about 75% sequence identity with wild-type humanalpha globin. However, a polypeptide of lesser sequence identity maystill be considered "substantially homologous" with alpha globin if ithas a greater sequence identity than would be expected from chance andalso has the characteristic higher structure of alpha globin and similarbiological activity. By way of comparison, Artemia's heme-bindingdomains are considered homologous with myoglobin even though the primarysequence similarity is no more than 27%, as alignment of theheme-binding domains around their conserved residues and the residuesconserved in other hemoglobins (i.e., involved in heme contacts or indetermining the relationship of the helical segments to each other)suggested that the Artemia domains possessed the classical globinhelices A to H with their corresponding turns, as well as variousconserved globin family residues. Also, among the serine proteaseinhibitors, there are families of proteins recognized to be homologousin which there are pairs of members with as little as 30% sequencehomology.

If the hemoglobin is to be produced by expression of recombinant DNA,the DNA can be engineered to produce desirable modified hemoglobins.Mutant hemoglobins can be advantageous when the hemoglobin is to be usedas a blood substitute without the benefit of the red blood cellenvironment. Certain mutant hemoglobins with high oxygen affinity wouldbe useful, for example, in delivery of oxygen to hypoxic tissues. Othermutants could be specifically designed to bind specific ligands otherthan oxygen for use in analytical assays or to scavenge and bind thenon-oxygen ligand from a solution. By applying the standard techniquesof site specific mutagenesis to the globin gene(s), (McCracken et al.,(1988) Biotechniques 6(4) 332-339 and Zoller et al., Methods inEnzymology 100; 468-500 (1987) are recent examples) one can add,subtract or change any amino acid or combination of amino acids in theresulting globin chain.

Well over a hundred mutants of human hemoglobin are known, affectingboth the alpha and beta chains, and the effect of many of thesemutations on oxygen-binding and other characteristics of hemoglobin areknown. Some preferred mutant hemoglobins include those disclosed in U.S.Pat. No. 5,028,588; PCT Patent Application WO 88/09179; PCT PatentApplication WO 90/13645; PCT Patent Application 93/08842; and U.S. Pat.No. 5,173,426. The human alpha and beta globins themselves differ at 84positions. In addition, interspecies variations in globin sequence havebeen extensively studied. Dickerson and Geis, (Hemoglobin Structure.Function, Evolution and Pathology, Benjamin Cummings Publishing Company,Menlo Park, Calif., (1983) Chapter 3) reported that in 1982, the 60known vertebrate alpha globins had identical residues at 23 of their 141positions, while for the 66 vertebrate beta globins considered, 20 ofthe 146 amino acids are identical. The 60 vertebrate myoglobins, whichalso belong to the globin family, had 27 invariant amino acids out of153 positions. If only mammals are considered, then the invariant aminoacids are 50/141 for the alpha globins, 51/146 for the beta globins, and71/153 for the myoglobins. Invariant positions cluster around thecenters of activity of the molecule: the heme crevice and theintersubunit contacts. Of the variable amino acids, some diverge fromthe consensus sequence for only a small fraction of the speciesconsidered.

The number of total differences between human alpha globin and selectedother vertebrate alpha globins is as follows: rhesus monkey (4), cow(17), platypus (39), chicken (35), human zeta (embryonic) (61), carp(71), and shark (88). For invertebrate globins the divergences are sealamprey (113), mollusc (124), Glycera (marine bloodworm) (124) andChironomus (midge) (131). Turning to the beta globin family, thedifferences of human beta globin from other vertebrate beta globins arerhesus monkey (8), human delta globin (10), cow beta globin (25), cowgamma globin (33), human gamma globin (39), human epsilon (embryonic)globin (36), platypus (34), chicken (45), shark (96), sea lamprey (123),mollusc (127), Glycera (125) and Chironomus (128).

Many of these differences may be misleading--variable amino acids mayexhibit only "conservative substitutions" of one amino acid for another,functionally equivalent one. A "conservative substitution" is asubstitution which does not abolish the ability of a globin-likepolypeptide (or domain) to incorporate heme and to associate with alphaand beta globin subunits to form a tetrameric (or pseudotetrameric)hemoglobin-like protein, which preferably will reversibly bind oxygen.The following resources may be used to identify conservativesubstitutions (and deletions or insertions):

(a) data on hemoglobin mutants (over a hundred such mutants exist);

(b) data on sequence variations among vertebrate, especially mammalian,alpha globins and beta globins;

(c) data on sequence variations among vertebrate, especially mammalian,myoglobins;

(d) data on sequence variations between vertebrate and invertebrateglobins, or among the invertebrate globins;

(e) data on the three-dimensional structures of human hemoglobin andother substantially homologous proteins, and molecular modellingsoftware for predicting the effect of sequence changes on suchstructures; and

(f) data on the frequencies of amino acid changes between members offamilies of homologous proteins (not limited to the globin family). See,e.g., Table 1-2 of Schulz and Schirmer, Principles of Protein Structure(Springer-Verlag: 1979) and FIG. 3-9 of Creighton, Proteins Structureand Molecular Properties (W. H. Freeman: 1983).

While the data from (a)-(d) is most useful in determining tolerablemutations at the site of variation in the cognate proteins, it may alsobe helpful in identifying tolerable mutations at analogous siteselsewhere in the molecule. Based on the data in category (f), thefollowing exchange groups may be identified, within which substitutionsof amino acids are frequently conservative

I. small aliphatic, nonpolar or slightly polar residues--Ala, Ser, Thr(Pro, Gly)

II. negatively charged residues and their amides--Asn Asp Glu Gln

III. positively charged residues--His Arg Lys

IV. large aliphatic nonpolar residues--Met Leu Ile Val (Cys)

V. large aromatic residues--Phe Tyr Trp

Three residues are parenthesized because of their special roles inprotein architecture. Gly is the only residue without a side chain andtherefore imparts flexibility to the chain. Pro has an unusual geometrywhich tightly constrains the chain. Cys can participate in disulfidebonds which hold proteins into a particular folding. Note that Schulzand Schimer would merge I and II above. Note also that Tyr, because ofits hydrogen bonding potential, has some kinship with Ser, Thr, etc.

In general, functionality is less likely to be affected by mutations atsurface residues, at least those not involved in either the heme creviceor the subunit contacts. In addition, "loops" connecting alpha helices,especially the D loop of the alpha helix, as well as free amino orcarboxy termini, are more tolerant of deletions and insertions.

Hemoglobin Ao is a heterotetramer composed of two alpha globin subunits(α₁,α₂) and two beta globin subunits (β₁, β₂). There is no sequencedifference between α₁ and α₂ or β₁ and β₂. The subunits arenoncovalently associated by Van der Waals forces, hydrogen bonds and,for deoxy Hgb, salt bridges. Hemoglobin is known to dissociate into α₁β₁ and α₂ β₂ dimers, which are eliminated from the bloodstream by renalfiltration. Intravascular retention of hemoglobin has been improved by,e.g., chemical crosslinking of subunits of a single tetramer, or betweentetramers.

As taught in U.S. Pat. No. 5,028,588 and PCT/US90/02654, it is possibleto produce a pseudotetrameric hemoglobin in which two noncovalentlyassociated subunits are replaced by a single pseudodimeric polypeptidewith two oxygen binding domains, joined either directly or by a linkerof one or more amino acids. This pseudodimeric polypeptide may beexpressed from a suitable fused gene. Thus, two alpha globin genes maybe fused into a "di-alpha globin" gene, or two beta globin genes into a"di-beta globin" gene, or alpha and beta globin genes into an "alphabeta" globin pseudodimer gene.

The advantage of fusing two or more globin chains together is that onecan selectively mutate one but not both of the chains, as taught inHoffman, et al., U.S. Ser. No. 789,179, filed Nov. 8, 1991, entitledProduction and Use of Hemoglobins and Analogues Thereof.

Hemoglobin has been modified using many techniques in the past. Any ofthese techniques may be used to prepare a hemoglobin component of theinvention that may be useful as a pharmaceutical composition. Examplesof such modifications are found in U.S. Pat. Nos. 4,412,989, 4,301,144,4,670,417, 4,321,259, 4,473,563, 4,710,488, 4,650,786, 4,336,248,4,598,064, 4,600,531, 4,377,512 and 5,173,426 among others. Individualglobin chains have been reasserted with modified forms to synthesize asemi-synthetic hemoglobin as well (Luisi et al., Nature (1986)320:555-556 and Nagai et al., Nature (1987) 329:858-860). Chemicallycrosslinked hemoglobins, or mutant hemoglobins which genetically fusethe alpha subunits (di-alpha Hgb) or the beta subunits (di-beta Hgb),may increase intravascular retention by inhibiting haptoglobin binding.Other modifications such as polymerization of globin chains,glycosylation, pegylation, encapsulation in a liposome or cell membranesare also contemplated.

Any of the above described hemoglobins may be used as starting materialsfor the method aspect fo the present invention.

Other proteins where N-terminal methylation can be decreased by themethod of the present invention include essentially any protein thatcontains an N-terminal methionine and a proline in position 4, when suchprotein is produced in a bacteria. These proteins can be obtained byreviewing any of a number of gene sequence and protein sequence databases including Genbank and Protein Identification Resources (NationalBiomedical Research Foundation, Washington, D.C.). Preferred proteinsfor use in the method aspect of this invention are those that have anN-terminal methionine, a leucine in position 2 and a proline in position4. A more preferred protein would be one that has an N-terminalmethionine, a leucine in position 2, a serine in position 3 and aproline in position 4. Preferred proteins with appropriate N-terminalsequence elements include vpu protein from human immunodeficiency virus(HIV), various proteins from various strains of human herpesvirus (e.g.,proteins, BFRF1, UL35, gene 41 protein, etc.), glyceraldehyde-3-phospatedehydrogenase, cyaB protein from Bordetella pertussis, various speciesof erythropoietin, carbon monoxide dehydrogenase, yeast alcoholdehydrogenase, thymidylate synthase and of course, L16 and CheZ.

To decrease or eliminate methylation of an N-terminal methionine of sucha protein, a proline at amino acid position 4 of said protein is mutatedto a non-proline residue. Mutation of an amino acid can be accomplishedby a number of methods that are known in the art. Mutation can occur ateither the amino acid level or at the codon level by altering thenucleotide sequence that codes for the amino acids. Mutation of aproline at position 4 can occur by substitution of another amino acidfor that proline. Mutation can also occur by adding one or more aminoacid at or prior to the proline at position 4 so as to shift the prolineto position 5 or higher. Mutation can also occur by deletion of one ormore amino acid prior to the proline at position 4 so as to shift theproline to position 3 or lower.

Substitution of an amino acid at position 4 of a protein can occur byaltering the codon that codes for that amino acid. This can beaccomplished by site directed mutagenesis using: (1) the Amershamtechnique (Amersham mutagenesis kit, Amersham, Inc., Cleveland, Ohio)based on the methods of Taylor et al., Nucl. Acids Res. (1985)13:8749-8764; Taylor et al., (1985) Nucl. Acids Res. 13:8764-8785;Nakamaye and Eskstein, (1986) Nucl. Acids Res. 14:9679-9698; and Denteet al., in DNA Cloning, Glover, Ed., IRL Press (1985) pages 791-802, (2)the Promega kit (Promega Inc., Madison, Wis.) or (3) the Biorad kit(Biorad Inc., Richmond, Calif.), based on the methods of Kunkel, (1985)Proc. Natl. Acad. Sci. USA 82:488; Kunkel et al., (1987) Meth. Enzymol.154:367; Kunkel, U.S. Pat. No. 4,873,192. It can also be accomplished byother commercially available or non-commercial means which incorporatethe technique of site-directed mutagenesis (usingmutant oligonucleotidesto achieve mutagenesis).

Site directed mutagenesis can also be accomplished using PCR basedmutagenesis such as that described in Zhengbin et al., pages 205-207 inPCR Methods and Applications, Cold Spring Harbor Laboratory Press, NewYork (1992); Jones and Howard, (1990) BioTechniques 8(2):178; Jones andHoward, (1991) BioTechniques 10:62-66.

Site directed mutagenesis can also be accomplished using casettemutagenesis with techniques that are known to those of skill in the art.

In addition to altering the codons that code for the amino acid atposition 4, chemical modification of the polypeptide sequence can beperformed.

The amino acid that is substituted for the proline at position 4 can beessentially any amino acid so long as the substitution does not have aneffect on the protein structure. Therefore, the substituted amino acidis preferably an uncharged amino acid, more preferably smaller unchargedamino acids such as glycine, valine, leucine, isoleucine, serine andthreonine, more preferably alanine, serine, threonine and valine, mostpreferably alanine and serine.

Addition of one or more amino acid to shift the proline at position 4 ofa protein to position 5 or higher can occur by adding the codons thatcode for the one or more amino acid. The amino acid or acids to be addedwill be added at or upstream of amino acid position 4. Such amino acidscan be added using the same methods as can be used to substitute adifferent amino acid for the proline at position 4. As with substitutionfor the proline at position 4, when an amino acid or acids is added ator upstream of amino acid position 4 to shift the proline at position 4of a protein to position 5 or higher, it is preferred that the addedamino acid or acids that are added is an uncharged amino acid, morepreferably smaller uncharged amino acids such as glycine, valine,leucine, isoleucine, serine and threonine, more preferably alanine,serine, threonine and valine, most preferably alanine and serine.Preferably, only one amino acid is added at or prior to the proline atposition 4.

Deletion of one or more amino acid to shift the proline at position 4 ofa protein to position 4 or lower can occur by deleting the codons thatcode for the one or more amino acid. The amino acid or acids to bedeleted will be deleted downstream of amino acid position 4. Such aminoacids can be deleted using the same methods as can be used to substitutea different amino acid for the proline at position 4. Preferably, onlyone amino acid is dleted at or prior to the proline at position 4.

A decrease in methylation of the N-terminal methionine can also beachieved by removing or decreasing the level of methionine at theN-terminal amino acid. If there is less methionine, there is lessmethylation of the N-terminal methionine, in fact, essentially nomethylation of the residual N-terminal methionine (see Table 4). Itappears that the amino acid in position 2 determines to a large extenthow much methionine excision will occur at the N-terminal amino acidwhen a protein is expressed in bacteria (Hirel, et al., (1989) Proc.Natl. Acad. Sci. USA 86:8247-8251). Preferably, the amino acid atposition 2 is mutated to an amino acid selected from the groupconsisting of glycine, alanine, proline, serine, threonine, valine andcysteine. Most preferably, the amino acid at position 2 is mutated to anamino acid selected from the group consisting of alanine, serine,threonine and valine.

The result of the method aspect of the present invention is a proteinwith decreased methylation compared to the methylation that would haveoccurred in the absence of the method of the present invention. Theprotein with a decreased methylation, or a demethylated protein, can beused for the therapeutic treatment of a disease but with less liklihoodof eliciting an immunological response. Further, the demethylatedprotein can be formulated into a pharmaceutical composition usingmethods and ingredients useful for the methylated protein.

The prevention of methylation of the N-terminal amino acid in arecombinant protein such as hemoglobin would be advantageous because ofthe immunogenic consequences that the methylation may imply. The use ofmost proteins, particularly recombinant proteins, is as therapeuticagents for the treatment and/or amelioration of disease or the symptomsassociated with a disease. Consequently, as they are administered to amammal, whether orally, intraveneously, subcutaneously or any otherroute, they eventually enter the bloodstream where any deviation in thestructure of the protein to that which is naturally occurring may elicitan immunogenic response. Therefore, modification of an N-terminalmethionine to a N-terminal-methyl methionine

All references cited herein are hereby incorporated by reference fortheir relevant teachings.

EXAMPLES

The following examples are provided by way of describing specificembodiments of the present invention without intending to limit thescope of the invention in any way.

EXAMPLE 1 Production of Protein Solution Containing Modified Hemoglobin

A. Construction of a Bacterial System for the Recombinant Production ofModified rHb1.1

Modified hemoglobins were produced by fermentation of the E. coli strain1661 carrying the plasmid pSGE705. Construction of pSGE705 is describedbelow.

Strain SGE661 carrying the plasmid pSGE705 is SGE1662.

Materials. pBR322, pUC19 and pNEB193 were purchased from New EnglandBiolabs, Beverly, Mass. Oligonucleotides were synthesized on an AppliedBiosystems DNA Synthesizer Model 392. The oligonucleotides used inpreparing pSGE705 are listed in Table 3. Restriction endonucleases werepurchased from New England Biolabs, Beverly, Mass. and used according tomanufacturer's specifications. T4 DNA Ligase was purchased from eitherNew England Biolabs, Beverly, Mass. or Gibco-BRL (Gaithersburg, Mass.)and used according to manufacturer's specifications. Pfu polymerase waspurchased from Stratagene (La Jolla, Calif.) and used according tomanufacturer's specifications.

Media used are described in J. H. Miller (Experiments in MolecularGenetics. Cold Spring Harbor Press, (1972) Cold Spring Harbor, N.Y.).and J. H. Miller (A Short Course in Bacterial Genetics. (1992) ColdSpring Harbor Press, Cold Spring Harbor, N.Y.). Acridine orange,ampicillin and kanamycin sulfate were purchased from Sigma Chemical Co.(St. Louis, Mo.). Tetracycline was purchased from Aldrich Chemicals(Milwaukee, Wis.).

Genetic and Molecular Biological Procedures. Standard bacterial geneticprocedures are described in J. H. Miller (Experiments in MolecularGenetics. (1972) Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) andJ. H. Miller (A Short Course in Bacterial Genetics. (1992) Cold SpringHarbor Press, Cold Spring Harbor, N.Y.). Standard molecular biologyprocedures were performed as described by Sambrook (Sambrook et al.,Molecular Cloning. (1989) Cold Spring Harbor Press, Cold Spring Harbor,N.Y.).

Plasmid DNA Transformation. DNA transformations were performed by theprocedure described by Wensick (Wensick et al., (1974) Cell 3:315-325).Briefly, cells were grown to mid log phase and then pelleted,resuspended in an equal volume of 10 mM MgSO₄ and incubated on ice for30 minutes. The cells were centrifuged and the pellet resuspended in 1/2original volume of 50 mM CaCl₂ and placed on ice for 20 minutes. Thecells were centrifuged again and then resuspended in 1/10 originalvolume of 50 mM CaCl₂. Plasmid DNA was added to the competent cells in asolution of 10 mM Tris-HCl pH 8.0, 10 mM MgCl₂ and 10 mM CaCl₂. Themixture was incubated on ice for 15 minutes and then incubated at 37° C.for 5 minutes. One milliliter of LB medium was added and the mixtureincubated with shaking for 30-60 minutes. The culture was thencentrifuged, resuspended in 0.1 ml of LB medium and plated on theappropriate selective medium.

Purification of DNA. DNA fragments were purified from an agarose gelusing the Geneclean system. (Bio 101, Inc. La Jolla, Calif.; methodprovided with product.) PCR products were prepared and cleaved withrestriction endonucleases using the Double Geneclean system. (Bio 101,Inc. La Jolla; method provided with product.) Briefly, the PCR productwas purified away from the PCR primers, then the PCR product was cleavedwith restriction endonuclease(s) and purified from the restrictionendonuclease and buffer. The PCR product was then ready for a ligationreaction.

                  TABLE 1                                                         ______________________________________                                        Plasmids                                                                        PLASMID   DESCRIPTION                                                       ______________________________________                                        pSGE1.1E4                                                                             rHb1.1 expression plasmid containing di-alpha and beta                   genes                                                                        pSGE1.1E5 like pSGE1.1E4 but ampicillin resistant instead of                   tetracycline resistant                                                       pSGE490 pUC19 lacI on a Bam HI-Hind III fragment                              pSGE491 pUC19 α on an Eco RI-Xba I fragment                             pSGE492 pNEB193 Ptac-α                                                  pSGE493 pUC19 β on an Xba I-Hind III fragment                            pSGE500 pUC19 α β on a Bam HI-Hind III fragment                    pSGE504 pSELECT-1 replace Sty I with a Pme I site                             pSGE505 pSGE504 rrnB T1 transcriptional terminator in the Eco                  RI-Cla I sites                                                               pSGE507 ColE1 ori and tet, 2213 bp                                            pSGE509 ColE1 ori tet lacI, 3425 bp                                           pSGE513 ColE1 ori tet lacI α β, 4386 bp                            pSGE515 ColE1 ori tet lacI diα β, 4812 bp                          pSGE700 pTZ18U + diα β from pSGE515                                pSGE705 modified rHb1.1 expression plasmid, ColE1 ori, tet, lacI,                     di-alpha and beta genes                                               pTZ18U a phagemid derivative of pUC19, for oligonucleotide                     directed mutagenesis                                                         pDLII-91F pGEM1 + α missing valine in 2nd position (Des-val)                   pNEB193 Like pUC19 but has more restriction sites in the multi                 cloning sites                                                         pBR322 ColE1 ori tet amp                                                      pRG1 pACYC177 lacIq                                                         ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    Oligonucleotides                                                              3') O  SEQUENCE (5'                                                                                        DESCRIPTION                                      __________________________________________________________________________    EV18   CGGGAATACGGTCTAGATCATTAA                                                                            C-term of α gene,                            SEQ. ID #8 CGGTATTTCGAAGTCAGAACG Xba I site                                   EV27 GATCCGAGCTGTTGACAATTAAT tac promoter                                     SEQ. ID #9 CATCGGCTCGTATAATGTGT sequence, Bam HI-                              GGAATTGTGACGGATAACAATTT Eag I sites                                           CACACAGGAAATTAATTAATGCT                                                       GTCTCC                                                                       EV28 GGCCGGAGACAGCATTAATTAAT tac promoter                                     SEQ. ID #10 TTCCTGTGTGAAATTGTTATCCGCTCAC sequence, Bam HI-                     AATTCCACACATTATACGAGCCGATGA Eag I sites,                                      TTAATTGTCAACAGCTCG complement of                                               EV27                                                                        EV29 TCGGATTCGAATTCCAAGCTGTTGG 5' end of α with                         SEQ. ID #11 ATCCTTAGATTGAACTGTCTCCGGCCG Eco RI, Bam HI and                     ATAAAACCACCG Eag I sites                                                     EV30 CGGAAGCCCAATCTAGAGGAA 5' end of β with                              SEQ. ID #12 ATAATATATGCACCTGACTCCG Xba I site                                  GAAGAAAAATCC                                                                 EV31 CCCGAAACCAAGCTTCATTAGTGA 3' end of the β                            SEQ. ID #13 GCTAGCGCGTTAGCAACACC gene with Hind III                             site                                                                        MW007 TTTAAGCTTCATTAGTGGTATT mutagenesis                                      SEQ. ID #14 TGTGAGCTAGCGCGT reverse primer                                      replaces last three                                                           codons of β                                                              missing in pSGE515                                                          MW008 CAGCATTAATTAACCTCCTTA mutagenesis                                       SEQ. ID #15 GTGAAATTGTTATCCG reverse primer                                     to optimize α                                                           ribozyme binding                                                              site (RBS)                                                                  MW009 GGTGCATATATTTACCTCCTT mutagenesis                                       SEQ. ID #16 ATCTAGATCATTAACGGTATTTCG reverse primer                             to optimize β RBS                                                        and remove second                                                             Bgl II site                                                                 TG14 GGTTTAAACC Pme I linker                                                  SEQ. ID #17                                                                   TG59 GGCGAATAAAAGCTTGCGGCCGCG Upstream of lacI                                SEQ. ID #18 TTGACACCATCGAATGGCGCAAAA gene, has a Hind III                      CCTTTCGCGG- and a Not I site                                                   upstream of the                                                               promoter                                                                    TG60 GGGCAAATAGGATCCAAAAAAAAG Downstream side                                 SEQ. ID #19 CCCGCTCATTAGGCGGGCTTTAT of lacI gene with                          CACTGCCCGCTTTCCAGTCGGG the trp                                                 transcriptional                                                               terminator and a                                                              Bam HI site                                                                 TG62 CCCCGAAAAGGATCCAAGTA upstream primer                                     SEQ. ID #20 GCCGGCGGCCGCGTTCCACTG for pBR322 ori                               AGCGTCAGACCCC positions 3170-3148                                              with a Bam HI and                                                             a Not I site                                                                TG63 GGCGGTCCTGTTTAAACGCT downstream                                          SEQ. ID #21 GCGCTCGGTCGTTCGGCTGCGG primer for pBR322                            ori positions                                                                 2380-2404 with                                                                a Pme I site                                                              __________________________________________________________________________

Annealing of oligonucleotides. Complementary oligonucleotides wereannealed according to the following procedure. Equimolar amounts of eacholigonucleotide were mixed in 15-25 μl of 10 mM Tris-HCl pH 8.0/1 mMEDTA and incubated at 65° C. for 30 minutes. The sample was transferredto a 37° C. water bath for 30 minutes. Finally, the sample was incubatedon ice for 60 minutes or in the refrigerator overnight.

Oligonucleotide directed mutagenesis. Oligonucleotide directedmutagenesis was performed with the Muta-gene phagemid in vitromutagenesis kit (Bio-Rad, Hercules, Calif.) according to manufacturer'sinstructions which are based on the method of Kunkel (Kunkel, T. A.(1985) Proc. Natl. Acad. Sci. USA 82:488; Kunkel et al., (1987) MethodsEnzymol. 154:367). The rHb1.1 region of pSGE515 was cloned into pTZ18U(Bio-Rad, Hercules, Calif. or U.S. Biochemical, Cleveland, Ohio) on aBam HI-Hind III fragment to create pSGE700. Three oligonucleotides,MW007, MW008 and MW009 were used to simultaneously introduce multiplechanges in a single reaction.

Preparation of pBR322 ori. PCR primers were designed to amplify thepBR322 origin of replication. These primers, TG62 and TG63, annealed tothe positions 2380-2404 and 3170-3148 on the pBR322 DNA sequence(Sutcliffe, J. G. 1979. Cold Spring Harbor Symp. Quant. Biol. 43:77-90).The PCR product was digested with Not I and Pme I. The DNA fragment waspurified according to the Geneclean procedure.

Preparation of tet gene fragment. The source for the tet gene waspSELECT-1 (Promega Corp., Madison, Wis.). This plasmid has a number ofrestriction endonuclease sites, such as Bam HI, Hind III, Sal I and SphI removed from the tet gene (Lewis and Thompson (1993) Nucleic AcidsRes. 18:3439-3443). A Pme I linker was inserted into the Sty I site ofpSELECT-1. This plasmid was designated pSGE504. Oligonucleotides TG71and TG72 were annealed and ligated to the Eco RI - Cla I fragment ofpSGE504. This plasmid, pSGE505, was shown to have the expectedrestriction endonuclease sites and to have lost the sites present in themulticloning site of pSELECT-1. pSGE505 was digested with Not I and PmeI. The 1417 bp fragment was purified according to the Genecleanprotocol.

Preparation of lacI gene. The lacI gene was isolated by amplifying thegene sequence from pRG1 (a gift from R. Garcia, Dana-Farber CancerInst., Boston) that carried the lacI gene. The PCR primers, TG59 andTG60 were designed to generate a wild type lacI promoter (Farabaugh, P.J. (1978) Nature 274:765), upstream of the gene and to place the trpterminator sequence (Christie et al., (1981) Proc. Natl. Acad. Sci. USA78:4180-4184) downstream of the gene. The same step could be carried outusing Y1089 (Promega) or chromosomal DNA from any E. coli straincarrying the lac region, such as MM294 (ATCC 33625.) The PCR product wasgel purified and isolated according to the Geneclean procedure andcloned into Bam HI-Hind III digested pUC19 DNA to make pSGE490.

Construction of pSGE515. PCR primers EV29 and EV18 were chosen toamplify the alpha gene from pDLII-91F (Hoffman et al., WO 90/13645). Thepurified PCR product was cleaved with the restriction endonucleases EagI and Xba I.

To create a plasmid that contained P_(tac) -α, the alpha gene (fromabove) and the tac promoter, which was prepared by annealing EV27 andEV28, were mixed with Eco RI-Xba I cleaved pUC19 DNA. The mixture of thethree DNA fragments, in approximately equimolar ratio, was treated withT4 DNA Ligase. After incubation the ligation mixture was used totransform SGE476 and ampicillin resistant transformants were selected.(Transformation into Strain MM294 (ATCC 33625) yields equivalentresults.) An isolate with the correct restriction endonuclease fragments(consistent with FIG. 1) was designated pSGE492. The α gene and the tacpromoter DNA sequences were verified by DNA sequencing.

Primers EV30 and EV31 were used to amplify the β gene from pSGE1.1E4 byPCR. The purified β gene fragment was digested with Xba I and Hind IIIand then mixed with Xba I-Hind III digested pUC19 DNA and treated withT4 DNA ligase. The ligation mixture was used to transform competentSGE476 (equivalent to MM294, ATCC 33625) and transformants were selectedon LB+ampicillin (100 μg/ml) plates. An isolate that contained theappropriate restriction endonuclease fragments (consistent with FIG. 1)was chosen and designated pSGE493. The β gene was confirmed by DNAsequencing.

The β gene was isolated from pSGE493 by restriction with Xba I andHindIII followed by purification according to the Geneclean method. ThisDNA fragment was then ligated to Xba I-Hind III restricted pSGE492 DNAand transformed into SGE713. (Any dam strain such as JM110 (ATCC 47013)or GM119 (ATCC 53339) could also be used.) An ampicillin resistanttransformant that carried a plasmid that had the appropriate restrictionfragments (consistent with FIG. 1) was chosen and designated pUC19αβ(pSGE500).

The Bam HI-Hind III fragment that contained the α and β genes of pSGE500was purified according to the Geneclean method. An Xho I fragment thatcarried a portion of the di-α gene containing the glycine linker regionwas gel purified from pSGE1.1E5. pSGE1.1E5 (described in Hoffman et al.,U.S. Ser. No. 789,179, filed Nov. 8, 1991) is a tetracycline sensitiveanalogue of pSGE1.1E4 (Hoffman et al., WO 90/13645), which could alsohave been used.

The pBR322 origin of replication region (pBR322 ori, above) was ligatedto the tet gene fragment (above) and the ligation mixture wastransformed into SGE476. (Transformation into MM294, above would yieldequivalent results.) Tetracycline resistant transformants were selectedand plasmid DNA was isolated and analyzed. An isolate that contained theappropriate restriction endonuclease fragments (consistent with FIG. 1)was chosen and designated pSGE507.

Next, pSGE507 and pSGE490 were digested with Bam HI and Not I and theappropriate fragments (consistent with FIG. 1) were purified. The twopurified fragments were ligated together and the ligation mixture wasused to transform competent SGE713. (Any dam strain could also be used;see above.) Tetracycline resistant transformants were selected, andplasmid DNA was isolated and analyzed. A plasmid that had theappropriate restriction fragments (consistent with FIG. 1) was chosenand designated pSGE509.

The purified Bam HI-Hind III fragment of pSGE500 that contained the αand β genes was ligated to Bam HI-Hind III digested pSGE509. Theligation mixture was used to transform pSGE713 (see above for equivalentstrains) and tetracycline resistant transformants were selected andcharacterized. An isolate yielding the correct size plasmid with theexpected restriction endonuclease fragments (consistent with FIG. 1) waschosen and designated pSGE513.

The Xho I fragment of pSGE1.1E5 (described in Hoffman et al., U.S. Ser.No. 789,179, filed Nov. 8, 1991) that contained the di-α glycine linkersequence was ligated to Xho I digested pSGE513 to create a plasmid thatcontained the di-α gene. SGE753 was transformed with the ligationmixture and tetracycline resistant transformants were selected.(Transformation into SGE800 would have yielded equivalent results.)Isolates were screened to identify those that contained the Xho Ifragment inserted into pSGE513 in the correct orientation (consistentwith FIG. 1). An isolate that contained the correct configuration of thedi-α gene, as determined by restriction endonuclease analysis with EagI, was designated pSGE515.

Modification of pSGE515 to create pSGE705. The DNA sequence record usedto design PCR primers for the amplification of the β gene did notcontain the C-terminal three amino acids. Oligonucleotide directedmutagenesis was used to add these nine nucleotides to the DNA sequenceof the β gene. In the same reactions, modifications were introduced tooptimize the ribosome binding sites for the di-α and β genes, and toremove a Bgl II site near the end of the di-α gene.

The following are the changes that were made with the oligonucleotidesMW008 and MW009 to optimize ribosomal binding sites and to remoe a BglIrestriction endonuclease site.

    __________________________________________________________________________    di alpha                                                                      __________________________________________________________________________    before - CAATTTCAC--AGGAAATTAATTAATGCTG                                                                 SEQ. ID #22                                                    ||||||.ve                              rtline.||**|.vertline                              .||**||.vert                              line.||||.ve                              rtline.||||.                              vertline.                                              after - CAATTTCACTAAGGAGGTTAATTAATGCTG SEQ. ID #23                         __________________________________________________________________________

Four nucleotide changes, shown above, including the insertion of twonucleotides, were introduced with MW008 to optimize the ribosome bindingsite for di-alpha. (|--indicates identity, *--indicates a change)

    __________________________________________________________________________    beta                                                                          __________________________________________________________________________    before - TAAaGATCTAGA---GGAAATAA-TATATGCAC                                                               SEQ. ID #24                                                   |||*|||.v                               ertline.|||.vertline                               .***|||**|.                               vertline.*|||.vertli                               ne.||||.ver                               tline.                                                after - TAATGATCTAGATAAGGAGGTAAATATATGCAC SEQ. ID #25                      __________________________________________________________________________

The six nucleotide changes shown above, including the insertion of fournucleotides, were introduced with MW009 to optimize the ribosome bindingsite for beta. The lower case "a" on the before strand was a T to Amutation in the construction of the alpha gene that introduced a Bgl IIsite into the sequence. This was removed so that there would only be asingle Bgl II site in pSGE705. (|)--indicates identity, *--indicates achange)

    ______________________________________                                        End of Beta                                                                   ______________________________________                                        before - CTCGCTCAC----------TAATGAA                                                                   SEQ. ID #26                                                      ||||||.ve                            rtline.||*********|.ver                            tline.||||.ver                            tline.                                                   after - CTCGCTCACAAATACCACTAATGAA SEQ. ID #27                              ______________________________________                                    

MW007 introduced the coding sequence for the last three amino acids ofthe beta gene as shown above. (|--indicates identity, *--indicates achange)

Putative mutants were screened for loss of a Bgl II restrictionendonuclease cleavage site (introduced by MW008). Seventeen of 24 hadlost the site and were further characterized by DNA sequencing at theother two mutagenized sites. One of the 17 had incorporated all threemodifications. These changes were verified by DNA sequencing and therHb1.1 genes were cloned into Bam HI-Hind III digested pSGE509. Anisolate that had the correct restriction endonuclease fragments wasdesignated pSGE705.

A plasmid map of pSGE705 is shown in FIG. 1. The plasmid map indicatesmany of the restriction endonuclease cleavage sites. pSGE705 is smallerthan its counterpart pSGE1.1E4, and the placement of its restrictionsites facilitates modular alterations of the sequence. An unusedantibiotic resistance marker was removed, and a promoter was added tothe lacI gene that would allow tighter control of rHb1.1 expression.

A new sequence upstream of the α gene minimized the distance between thetac promoter (De Boer et al., (1983) Proc. Natl. Acad. Sci. USA80:21-25) and the first codon of the alpha gene. The intergenic regionbetween the di-α gene and the β gene was also designed to contain theminimum sequence that contained a restriction endonuclease site and theribosome binding site for the β gene.

On Jan. 20, 1994 E. coli strain SGE1661 was deposited with the AmericanType Culture Collection (ATCC Accession Number 55545) under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and the Regulationsthereunder (Budapest Treaty). This assures maintenance of a viableculture for 30 years from the date of deposit. The organisms will bemade available by the ATCC under the terms of the Budapest Treaty, andsubject to an agreement between Applicants and ATCC which assuresunrestricted availability upon issuance of the perinent U.S. patent.Availability of deposited strains is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

B. Fermentations

Fermentor Inoculum (500 mL broth in 2 L shake flasks)

To prepare the fermentor inoculum, seed stock was thawed. Seed stock(100μl) was grown up in 500 ml of DM1 in an Erlenmeyer flask at 37° C.in s 1 inch rotary shaker (275 to 300 rpm) for 8 to 10 hours. DM1 mediais:

4.1 g/L KH₂ PO₄

7.0 g/L K₂ HPO₄

2.0 g/L (NH₄)₂ SO₄

1.0 g/L Na₃ Citrate·2H₂ O

153 mg/L MgSO₄,·7H₂ O

up to 2.30 g/L of L-proline,

2.5 mL/L of a trace metal solution containing:

32.5 μg/ml FeCl₃ ·6H₂ O

1.56 μg/ml ZnCl₂

2.4 μg/ml CoCl₂ ·6H₂ O, 2.4 μg/ml Na₂ MoO₄ ·2H₂ O,

1.22 mg/mL CaCl₂ ·2H₂ O,

1.54 μg/ml Cu(II)SO₄ ·5H₂ O,

0.6 μg/ml H₃ BO₃,

120 μl/ml HCl dissolved in purified water

After sterilization of the above solution, the following components wereadded to achieve the final concentrations indicated:

20 mL/L 10% yeast extract/L

4.0 mL 60% glucose solution/L

0.06 mg/L of sterile-filtered 125 mg/mL thiamine HCl dissolved inpurified water

0.1 mg/L of tetracycline in an ˜50% ethanol solution

Fermentor (2 L volume)

200 mL of the Fermentor Inoculum was then asceptically transferred to a2-liter New Brunswick fermentor containing 1800 mL of a solutioncontaining:

1.83 g/L KH₂ PO₄

3.27 g/L K₂ HPO₄

1.83 g/L (NH₄)₂ SO₄

After sterilization of the above solution, the following components wereadded to achieve the final concentrations indicated:

1.36 g/L TriSodium Citrate

1.36 g/L MgSO₄ ·7H₂ O

2.87 g/L proline

3.05 g/L of the Trace Metal solution described above

0.1 mg/L tetracycline in 50% ethanol solution,

0.06 mg/L thiamine HCl in purified water, sterile filtered solution

200 g/L of 70% glucose

50+10 g/L of 30% NH₄ OH

2 ml PPG 2000

The fermentor is run at 30±1° C., controlling dissolved oxygen at 20%and glucose between 0-6 g/L. At OD 30±2, induction occurs by adding 1.4mL of 100 mM Isopropyl thiogalactoside (IPTG) and 1.5 mL of 50 mg/mLhemin. At 3 hours post induction, 2.0 mL of 50 mg/mL hemin is added andat 6 hours post induction, 2.5 mL of 50 mg/mL hemin is added. Harvestand further purification occurs at 10 hours post induction.

C. Purification

Frozen cells were partially thawed in warm water for approximately 20-30minutes. Cells were chopped into small bits in a steel beaker usingbreak buffer (40 mM Tris base, 1 mM benzamidine) as needed. The choppedcells and break buffer at a ratio of 2 mL break buffer per 1 gram offrozen cells were placed in a Waring Industrial Blender and homogenizedfor 3-5 minutes on the low setting. The solution was allowed to settlefor 5 minutes after homogenization and any foamed material was removed.

A Niro Panda™ cell disruption device (Niro Hudson, Inc. Hudson, Wis.)was used for homogenization by passing 200 mL of buffer through thesystem. Cells were lysed by two passages of the homogenized cellsolution through the Niro set at 850 bar. The pH of the lysate wasadjusted to approximately 8 with sodium hydroxide, and sufficientZn(OAc)₂ was added to make the solution 2 mM in Zn(OAc)₂. The solutionwas then spun at 10,000 rpm in a JA-10 rotor at 4° C. for 60 minutes ina Beckman centrifuge. The supernatant was collected and diluted 1:1 withdistilled water.

Chromatography

All solutions were 4° C. and were adjusted to the correct pH at 4° C.500 mL of Chelating Sepharose fast flow resin (Pharmacia, Piscataway,N.J.) was prepared by washing with 4 column volumes of distilled water.Flow through the column for all steps was 200 mL/min. The resin wascharged with 2 to 3 column volumes of 2 mM Zn(OAc)₂ followed by 2-3column volumes of 200 mM NaCl. The lysate was loaded onto the column andwashed with 4 to 6 column volumes of 20 mM Tris, 500 mM NaCl, pH 8.5,7-8 column volumes of 240 mM Tris, pH 8.5, and 7-8 column volumes of 20mM Tris, pH 8.5. Hemoglobin was eluted with 15 mM EDTA, 20 mM Tris, pH8.5 and collected into 200 mL of well oxygenated 20 mM Tris, pH 8.5. Thecolumn was then rinsed with an additional 3-4 column volumes of 15 mMEDTA, 20 mM Tris, pH 8.5, regenerated with 4 column volumes of 200 mMNaCl and stored in 0.2 N NaOH.

The solution was then buffer exchange 5 times into 20 mM Tris, pH 8.5prior to loading onto 200 mL of a Sepharose Q column. The column hadbeen prepared by rinsing with 4 column volumes of distilled water, 4column volumes of 1 M NaCl, 4 additional column volumes of distilledwater and equilibrating with 3 to 4 column volumes of 20 mM Tris, pH8.5. After loading the sample, the column was washed with 2 to 3 columnvolumes of 20 mM Tris, pH 8.5 and eluted with 20 mM Tris, pH 7.6.Fractions were collected and pooled if the A₅₇₅ /A₅₄₀ ratio was greaterthan or equal to 1.03. The column was then cleaned with 3-4 columnvolumes of 1 M NaCl, 4 column volumes of distilled water, 2-3 columnvolumes of 50% acetic acid, 4 column volumes of distilled water andfinally 2-3 column volumes of 0.2 N NaOH for storage. The column was runat 30 mL/min flow rate. The resultant hemoglobin was stored at -80° C.or in liquid nitrogen.

EXAMPLE 2 Construction of Hemoglobin Mutants with Substitution forProline at Position 4

The rHb1.1 expression cassette was subcloned into the phagemid vectorpTZ19U as a BamHI-PstI DNA fragment. This phagemid vector allowsproduction of both single and double-stranded DNA. The phagemid alsocontains the Amp resistance gene for selection of transformants in E.coli.

This phagemid (called pTZ19U/705) was transformed into E. coli strainTG1 (Amersham, Cleveland, Ohio) and single-stranded DNA was isolatedaccording to the protocol described in the Amersham mutagenesis kit withthe exception that additional chloroform, phenol/chloroform andchloroform/isoamyl alcohol extractions were incorporated in order toobtain very clean DNA.

The oligonucleotides were gel purified on 20% acrylamide gels prior touse in the mutagenesis procedure. The oligonucleotides used inconstruction of the various mutants are listed below, along with thecorresponding amino acid sequence. The mutation is underlined in eachcase.

    5'GGA-GGT-TAA-TTA-ATG-CTG-TCT-GCC-GCC-GAT-AAA              (SEQ. ID. NO. 28)

    MET-LEU-SER-ALA-ALA-ASP-LYS                                (SEQ. ID. NO. 29)

Mutagenesis of rHb1.1 using each of the above oligonucleotides, wascarried out according to the protocol outlined in the Amershammutagenesis kit. The final ligation mixture containing the mutant DNAwas transformed into strain TG1 and plated on LB/Amp plates. Doublestranded DNA from individual transformant colonies was digested withBamH1-Pst1 to confirm that the rHb1.1 gene cassette was still in themutant phagemids, then double stranded DNA from several mutant colonieswas sequenced to confirm the presence of the mutation using theSequenase kit (USB). DNA from each mutant was digested with BamHI-PstIand the rHb1.1 DNA fragment was isolated and subcloned into theexpression vector pSGE509. These subclones were transformed into strainSGE1661 and selected on LB/Tet plates. Individual transformants werestreak-isolated, prior to isolation of DNA and subsequent sequencing ofthe dialpha and beta genes. The mutants were tested for induction ofhemoglobin before fermentation and purification of the mutanthemoglobins. Table 3 shows the extent of Met processing (percentage ofMet that was cleaved or removed from the protein), extent of N-Metmethylation, and activity data (as measured by P50 and Hill coefficients(nmax) for a number of amino acid additions prior to the naturallyoccurring proline at position 5 in the recombinant hemoglobin rHb1.1.

                  TABLE 3                                                         ______________________________________                                                      MET            FUNCTIONAL                                         MUTATION PROCESSING DATA                                                    ______________________________________                                        Met-Leu-Ser-Ala                                                                             no Met removal,                                                                              P.sub.50 = 31.72                                   (SEQ. ID. NO. 30) no N-methyl Met n.sub.max = 1.79                            repeat fermentation no Met removal,                                            no N-methyl Met                                                              Met-Leu-Ser-Ser  no Met removal, P.sub.50 = 32.34                             (SEQ. ID. NO. 31) no N-methyl Met n.sub.max = 1.61                            control fermentation no Met removal,                                           32% methyl Met                                                               reference rHb1.1 no Met removal, P.sub.50 = 31.78                              30-40% methyl Met n.sub.max = 2.12                                         ______________________________________                                    

EXAMPLE 3 Construction of Hemoglobin Mutants with Amino Acid Addition toShift Proline from Position 4

The rHb1.1 expression cassette was subcloned into the phagemid vectorpTZ19U as a BamHI-PstI DNA fragment. This phagemid vector allowsproduction of both single and double-stranded DNA. The phagemid alsocontains the Amp resistance gene for selection of transformants in E.coli.

This phagemid (called pTZ19U/705) was transformed into E. coli strainTG1 and single-stranded DNA was isolated according to the protocoldescribed in the Amersham mutagenesis kit with the exception thatadditional chloroform, phenol/chloroform and chloroform/isoamyl alcoholextractions were incorporated in order to obtain very clean DNA.

The oligonucleotides were gel purified on 20% acrylamide gels prior touse in the mutagenesis procedure. The oligonucleotides used inconstruction of the various mutants are listed below, along with thecorresponding amino acid sequence. The mutation is underlined in eachcase.

    5'GGA-GGT-TAA-TTA-ATG-GCC-CTG-TCT-CCG                      (SEQ. ID. NO. 32)

    MET-ALA-LEU-SER-PRO                                        (SEQ. ID. NO. 33)

    5'GGA-GGT-TAA-TTA-ATG-TCT-CTG-TCT-CCG                      (SEQ. ID. NO. 34)

    MET-SER-LEU-SER-PRO                                        (SEQ. ID. NO. 35)

    5'GGA-GGT-TAA-TTA-ATG-ACC-CTG-TCT-CCG                      (SEQ. ID. NO. 36)

    MET-THR-LEU-SER-PRO                                        (SEQ. ID. NO. 37)

    5'GGA-GGT-TAA-TTA-ATG-GTT-CTG-TCT-CCG                      (SEQ. ID. NO. 38)

    MET-VAL-LEU-SER-PRO                                        (SEQ. ID. NO. 39)

    5'GGA-GGT-TAA-TTA-ATG-GTT-CTG-TCT-GCC                      (SEQ. ID. NO. 40)

    MET-VAL-LEU-SER-ALA                                        (SEQ. ID. NO. 41)

Mutagenesis of rHb1.1 using each of the above oligonucleotides, wascarried out according to the protocol outlined in the Amershammutagenesis kit. The final ligation mixture containing the mutant DNAwas transformed into strain TG1 and plated on LB/Amp plates. Doublestranded DNA from individual transformant colonies was digested withBamH1-Pst1 to confirm that the rHb1.1 gene cassette was still in themutant phagemids, then double stranded DNA from several mutant colonieswas sequenced to confirm the presence of the mutation using theSequenase kit (USB). DNA from each mutant was digested with BamHI-PstIand the rHb1.1 DNA fragment was isolated and subcloned into theexpression vector pSGE509. These subclones were transformed into strainSGE1661 and selected on LB/Tet plates. Individual transformants werestreak-isolated, prior to isolation of DNA and subsequent sequencing ofthe dialpha and beta genes. The mutants were tested for induction ofhemoglobin before fermentation and purification of the mutanthemoglobins. Table 4 shows the extent of Met processing (percentage ofMet that was cleaved or removed from the protein), extent of N-Metmethylation, and activity data (as measured by P50 and Hill coefficients(nmax) for a number of amino acid additions prior to the naturallyoccurring proline at position 5 in the recombinant hemoglobin rHb1.1.

                  TABLE 4                                                         ______________________________________                                                     MET            FUNCTIONAL                                          MUTATION PROCESSING DATA                                                    ______________________________________                                        Met-Ala-Leu-Ser-Pro                                                                        52%                                                                (SEQ. ID. NO. 33) no N-methyl Met                                             Met-Ser-Leu-Ser-Pro 75% (by sequencing) P.sub.50 = 27.59                      (SEQ. ID. NO. 35) 90-100% (by ESMS) n.sub.max = 1.75                           no N-methyl Met                                                            Leu-Ser-Pro 44% P.sub.50 = 32.44                                                (SEQ. ID. NO. 37) no N-methyl Met n.sub.max = 1.91                          Leu-Ser-Pro 17% p.sub.50 = 26.7                                                 (SEQ. ID. NO. 39) no N-methyl Met n.sub.max = 1.9                           Leu-Ser-Ala   28% P                                                                        .sub.50 = 33.27                                                    (SEQ. ID. NO. 41) no N-methyl Met n.sub.max = 1.48                          Alaet-Leu-Ser no Met removal, P                                                            .sub.50 = 31.72                                                    (SEQ. ID. NO. 29) no N-methyl Met n.sub.max = 1.79                            repeat fermentation no Met removal,                                            no N-methyl Met                                                              Met-Leu-Ser-Ser            no Met removal, P                                                            .sub.50 = 32.34                                     (SEQ. ID. NO. 42) no N-methyl Met n.sub.max = 1.61                            control fermentation no Met removal,                                           32% methyl Met                                                               reference rHb1.1 no Met removal, P.sub.50 = 31.78                              30-40% methyl Met n.sub.max = 2.12                                         ______________________________________                                    

EXAMPLE 4 Construction of CheZ Mutants with Substitution for Proline atPosition 4

The CheZ expression cassette is subcloned into the phagemid vectorpTZ19U as a BamHI-PstI DNA fragment. This phagemid vector allowsproduction of both single and double-stranded DNA. The phagemid alsocontains the Amp resistance gene for selection of transformants in E.coli.

This phagemid (called pTZ19U/805) is transformed into E. coli strain TG1and single-stranded DNA is isolated according to the protocol describedin the Amersham mutagenesis kit with the exception that additionalchloroform, phenol/chloroform and chloroform/isoamyl alcohol extractionsare incorporated in order to obtain very dean DNA.

The oligonucleotides are gel purified on 20% acrylamide gels prior touse in the mutagenesis procedure. The oligonucleotide used inconstruction of the various mutants is listed below, along with thecorresponding amino acid sequence. The mutation is underlined.

    5'-GGA-GGT-TAA-TTA-ATG-CTG-TCT-GCC-GCC-GAT-AAA             (SEQ. ID. NO. 28)

    MET-LEU-SER-ALA-ALA-ASP-LYS                                (SEQ. ID. NO. 29)

Mutagenesis of CheZ using each of the above oligonucleotides, is carriedout according to the protocol outlined in the Amersham mutagenesis kit.The final ligation mixture containing the mutant DNA is transformed intostrain TG1 and plated on LB/Amp plates. Double stranded DNA fromindividual transformant colonies is digested with BamH1-Pst1 to confirmthat the CheZ gene cassette is still in the mutant phagemids, thendouble stranded DNA from several mutant colonies is sequenced to confirmthe presence of the mutation using the Sequenase kit (USB). DNA fromeach mutant is digested with BamHI-PstI and the CheZ DNA fragment isisolated and subcloned into the expression vector pSGE509. Thesesubclones are transformed into strain SGE1661 and selected on LB/Tetplates. Individual transformants are streak-isolated, prior to isolationof DNA and subsequent sequencing of the dialpha and beta genes. Themutants are tested for induction of CheZ before fermentation andpurification of the mutant CheZ.

EXAMPLE 5 Construction of L16 Mutants with Substitution for Proline atPosition 4

The L16 expression cassette is subcloned into the phagemid vector pTZ19Uas a BamHI-PstI DNA fragment. This phagemid vector allows production ofboth single and double-stranded DNA. The phagemid also contains the Ampresistance gene for selection of transformants in E. coli.

This phagemid (called pTZ19U/805) is transformed into E. coli strain TG1and single-stranded DNA is isolated according to the protocol describedin the Amersham mutagenesis kit with the exception that additionalchloroform, phenol/chloroform and chloroform/isoamyl alcohol extractionsare incorporated in order to obtain very dean DNA.

The oligonucleotides are gel purified on 20% acrylamide gels prior touse in the mutagenesis procedure. The oligonucleotide used inconstruction of the various mutants is listed below, along with thecorresponding amino acid sequence. The mutation is underlined.

    5'-GGA-GGT-TAA-TTA-ATG-CTG-TCT-GCC-GCC-GAT-AAA             (SEQ. ID. NO. 28)

    MET-LEU-SER-ALA-ALA-ASP-LYS                                (SEQ. ID. NO. 29)

Mutagenesis of L16 using each of the above oligonucleotides, is carriedout according to the protocol outlined in the Amersham mutagenesis kit.The final ligation mixture containing the mutant DNA is transformed intostrain TG1 and plated on LB/Amp plates. Double stranded DNA fromindividual transformant colonies is digested with BamH1-Pst1 to confirmthat the L16 gene cassette is still in the mutant phagemids, then doublestranded DNA from several mutant colonies is sequenced to confirm thepresence of the mutation using the Sequenase kit (USB). DNA from eachmutant is digested with BamHI-PstI and the L16 DNA fragment is isolatedand subcloned into the expression vector pSGE509. These subclones aretransformed into strain SGE1661 and selected on LB/Tet plates.Individual transformants are streak-isolated, prior to isolation of DNAand subsequent sequencing of the dialpha and beta genes. The mutants aretested for induction of L16 before fermentation and purification of themutant L16.

EXAMPLE 6 Construction of CheZ Mutants with Addition to Shift Prolinefrom Position 4

The CheZ expression cassette is subcloned into the phagemid vectorpTZ19U as a BamHI-PstI DNA fragment. This phagemid vector allowsproduction of both single and double-stranded DNA. The phagemid alsocontains the Amp resistance gene for selection of transformants in E.coli.

This phagemid (called pTZ19U/805) is transformed into E. coli strain TG1and single-stranded DNA was isolated according to the protocol describedin the Amersham mutagenesis kit with the exception that additionalchloroform, phenol/chloroform and chloroform/isoamyl alcohol extractionswere incorporated in order to obtain very dean DNA.

The oligonucleotides are gel purified on 20% acrylamide gels prior touse in the mutagenesis procedure. The oligonucleotides used inconstruction of the various mutants are listed below, along with thecorresponding amino acid sequence. The mutation is underlined in eachcase.

    5'-GGA-GGT-TAA-TTA-ATG-GCC-CTG-TCT-CCG                     (SEQ. ID. NO. 32)

    MET-ALA-LEU-SER-PRO                                        (SEQ. ID. NO. 33)

    5'-GGA-GGT-TAA-TTA-ATG-TCT-CTG-TCT-CCG                     (SEQ. ID. NO. 34)

    MET-SER-LEU-SER-PRO                                        (SEQ. ID. NO. 35)

    5'-GGA-GGT-TAA-TTA-ATG-ACC-CTG-TCT-CCG                     (SEQ. ID. NO. 36)

    MET-THR-LEU-SER-PRO                                        (SEQ. ID. NO. 37)

    5'-GGA-GGT-TAA-TTA-ATG-GTT-CTG-TCT-CCG                     (SEQ. ID. NO. 38)

    MET-VAL-LEU-SER-PRO                                        (SEQ. ID. NO. 39)

    5'-GGA-GGT-TAA-TTA-ATG-GTT-CTG-TCT-GCC                     (SEQ. ID. NO. 40)

    MET-VAL-LEU-SER-ALA                                        (SEQ. ID. NO. 41)

Mutagenesis of CheZ using each of the above oligonucleotides, is carriedout according to the protocol outlined in the Amersham mutagenesis kit.The final ligation mixture containing the mutant DNA is transformed intostrain TG1 and plated on LB/Amp plates. Double stranded DNA fromindividual transformant colonies is digested with BamH1-Pst1 to confirmthat the CheZ gene cassette was still in the mutant phagemids, thendouble stranded DNA from several mutant colonies is sequenced to confirmthe presence of the mutation using the Sequenase kit (USB). DNA fromeach mutant is digested with BamHI-PstI and the CheZ DNA fragment isisolated and subcloned into the expression vector pSGE509. Thesesubclones are transformed into strain SGE1661 and selected on LB/Tetplates. Individual transformants are streak-isolated, prior to isolationof DNA and subsequent sequencing of the dialpha and beta genes. Themutants are tested for induction of CheZ before fermentation andpurification of the mutant CheZ.

EXAMPLE 7 Construction of L16 Mutants with Addition to Shift Prolinefrom Position 4

The L16 expression cassette is subcloned into the phagemid vector pTZ19Uas a BamHI-PstI DNA fragment. This phagemid vector allows production ofboth single and double-stranded DNA. The phagemid also contains the Ampresistance gene for selection of transformants in E. coli.

This phagemid (called pTZ19U/905) is transformed into E. coli strain TG1and single-stranded DNA was isolated according to the protocol describedin the Amersham mutagenesis kit with the exception that additionalchloroform, phenol/chloroform and chloroform/isoamyl alcohol extractionswere incorporated in order to obtain very clean DNA.

The oligonucleotides are gel purified on 20% acrylamide gels prior touse in the mutagenesis procedure. The oligonucleotides used inconstruction of the various mutants are listed below, along with thecorresponding amino acid sequence. The mutation is underlined in eachcase.

    5'-GGA-GGT-TAA-TTA-ATG-GCC-CTG-TCT-CCG                     (SEQ. ID. NO. 32)

    MET-ALA-LEU-SER-PRO                                        (SEQ. ID. NO. 33)

    5'-GGA-GGT-TAA-TTA-ATG-TCT-CTG-TCT-CCG                     (SEQ. ID. NO. 34)

    MET-SER-LEU-SER-PRO                                        (SEQ. ID. NO. 35)

    5'-GGA-GGT-TAA-TTA-ATG-ACC-CTG-TCT-CCG                     (SEQ. ID. NO. 36)

    MET-THR-LEU-SER-PRO                                        (SEQ. ID. NO. 37)

    5'-GGA-GGT-TAA-TTA-ATG-GTT-CTG-TCT-CCG                     (SEQ. ID. NO. 38)

    MET-VAL-LEU-SER-PRO                                        (SEQ. ID. NO. 39)

    5'-GGA-GGT-TAA-TTA-ATG-GTT-CTG-TCT-GCC                     (SEQ. ID. NO. 40)

    MET-VAL-LEU-SER-ALA                                        (SEQ. ID. NO. 41)

Mutagenesis of L16 using each of the above oligonucleotides, is carriedout according to the protocol outlined in the Amersham mutagenesis kit.The final ligation mixture containing the mutant DNA is transformed intostrain TG1 and plated on LB/Amp plates. Double stranded DNA fromindividual transformant colonies is digested with BamH1-Pst1 to confirmthat the L16 gene cassette was still in the mutant phagemids, thendouble stranded DNA from several mutant colonies is sequenced to confirmthe presence of the mutation using the Sequenase kit (USB). DNA fromeach mutant is digested with BamHI-PstI and the L16 DNA fragment isisolated and subcloned into the expression vector pSGE509. Thesesubclones are transformed into strain SGE1661 and selected on LB/Tetplates. Individual transformants are streak-isolated, prior to isolationof DNA and subsequent sequencing of the dialpha and beta genes. Themutants are tested for induction of L16 before fermentation andpurification of the mutant L16.

EXAMPLE 8 Measurement of Methylation of the N-terminal Amino AcidSignaled by Presence of Proline in Position 4

The level of methylation was obtained by sequencing proteins, In thecase of di-alpha hemoglobin (rHb1.1 and mutants thereof), the dialphachain was separated prior to sequencing. Since PTH-N-methyl methionineand PTH-Isoleucine essentially coelute, it was assumed that the colorfactor for PTH-N-methyl methionine and PTH-Isoleucine were equal. Bycomparing the yields of PTH-N-methyl methionine and PTH-Methionine, itwas possible to calculte the level of methylation.

Additionally, to estimate the level of methylation in the different lotsof the proteins, V8 mapping was used. In the case of the dialphahemoglobin (rHb1.1 and metants thereof), the dialpha chain wasmaintained intact for this procedure. The area under the peaks K and Lwere compared wherein peak K was the unmethylated N-terminal peptideMet₁ - . . . -Glu₃₀ of the dialpha chain and L is the methylatedderivative of the same peptide. Table 5 shows a comparison of themeasurement of methylation by the two methods for four differentfermentation runs of unmodified rHb1.1. Further descriptions of the twomeasurement techniques used are provided below.

                  TABLE 5                                                         ______________________________________                                        Lot #      by Sequencing [%]                                                                          by Mapping [%]                                        ______________________________________                                        A          33.4         41.3                                                      41.9                                                                        B 34.8 40.8                                                                     35.5                                                                        C 32.0 43.7                                                                     37.7                                                                        D 26.4 42.9                                                                     40.0                                                                      ______________________________________                                    

Isolation and Alkylation of Chains

The globins from 1 ml of concentrated rHb1.1 (50 mg/ml) wereprecipitated with 20 volumes of cold acetone containing 0.2% HCl.Soluble heme remained in solution. The precipitated globins were spundown and washed 2 times with 5 ml of the same cold acetone. Theheme-free globins were dissolved in 2% formic acid and put on SEC column(Pharmacia S-100, RH16-50) running with 2% formic acid at 0.5 ml/min.Pooled fractions of separated chains containing about 15 mg of proteinof each chain were collected and lypholized. About 6 mg of eachlyophilized isolated chain was dissolved in 250 μl of solutioncontaining 100 mM Tris buffer pH 8.5, 6 M guanidine hydrochloride, and50 mM dithiothreitol (DTT). The mixtures were incubated under argon at37° C. for 1 hour. After cooling, the reduced cyteines were alkylated byaddition of 3 μl of vinylpyridine and reacted at room temperature for 1hour. The alkylated globins were purified on SEC (S-100) as describedabove.

Peptide Mapping

S. aureus V-8 protease, purchased from Pierce, Rockford, Ill., was usedto map recombinant hemoglobin. About 1 mg of alkylated and purifiedchains were dissolved in 100 μl of 8 M urea and diluted to 400 μl with100 mM ammonium acetate pH 4.0. The condition for this digest was a 1:30(w/w) enzyme:substrate ratio and overnight digestion at roomtemperature. In the morning, and extra amount of V-8 was added to bringthe final ration to 1:25 and the solution was incubated for anadditional two hours. The digests were then chromatographed on a 4.6×250mm Vydac C₄ column (HP 1090 Series 2 liquid chromatograph, HewlettPackard, Palo Alto, Calif.) Solvent A was 0.1% (v/v) trifluoroaceticacid (TFA) and solvent B was 70% acetonitrile with 0.1% (v/v) TFA (flow1 ml/min, start with 5% B, hold 5% B for 5 min, then 5% to 70% in 65min). The chromatography was monitored at 215 and 400 nm.

Analytical C₄ Separation

Recombinant hemoglobin (about 100 μg) was separated on the Vydac C₄analytical column (0.46×35 cm) in the gradient of acetonitrile. SolventA was 20% acetonitrile with 0.1 % TFA and solvent B was 70% acetonitrilewith 0.1 % TFA (flow 1 ml/min, start with 30% B, hold for 5 min, then30% to 70% in 65 min).

Sequencing

Proteins and peptides were sequenced by automated Edman degradationchemistry on the Porton 2090E gas phase sequencer (Beckman Instruemnts,Fullerton, Calif.). Porton (Beckman Instruments, Fullerton, Calif.)supports were used. PTH-amino acids (from Pierce, Rockford, Ill.) wereidentified (by reverse phase chromatography on a modifiedHewlett-Packard 1090L HPLC using a H.P. AminoQuant column(Hewlett-Packard, Palo Alto, Calif.)). The data was analyzed on anEverex 286 computer (Everex Systems, Inc., Fremont, Calif.) using Portonchromatography software (developed by Everex Inc., Fremont, Calif.).

Electro Spray Mass Spectrometry

Mass spectrometry was used to determine the masses of mutants ofrecombinant hemoglobin and separated chains. Electrospray mass spectrawere obtained using a Vestec electrospray source and a model 201 singlequadropole mass spectrometer with a 2000 AMU range (Vestec, Houston,Tex.). Samples were deliveered to the source at 1.6 μl/min at aconcentration below 0.1 mg/ml in organic mixture. When experiments wereperformed on the entire recombinant hemoglobin, starting material wasdiluted at least 100 times with the mixture containing 1:1water:acetonitrile and 3% acetic acid or in some experiments 1:1 mixtureof water/methanol and 1% acetic acid was used. Similar concentrationwere used for separated chains. Other operating conditions were asrecommended by the manufacturer (Vestec, Houston, Tex.). Horse heartmyoglobin at concentration 0.02 mg/ml (M.W. 16950.6) from Sigma (St.Louis, Mo.) has been used daily to calibrate the instruemnt.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 42                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Escherichia coli                                              <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: METHYLATION- N terminal M - #et                       - - <400> SEQUENCE: 1                                                         - - Met Leu Gln Pro                                                            1                                                                            - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Escherichia coli                                              <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: METHYLATION-  N terminal - #Met                       - - <400> SEQUENCE: 2                                                         - - Met Met Gln Pro                                                            1                                                                            - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: L-16               peptide                                                                  - - <400> SEQUENCE: 3                                                         - - Met Leu Ser Pro                                                            1                                                                            - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: rHb1.1              peptide                                                                  - - <400> SEQUENCE: 4                                                         - - Met Leu Ser Pro                                                            1                                                                            - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: METHYLATION- N terminal M - #et                      <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: rhb1.1              dialpha peptide                                                          - - <400> SEQUENCE: 5                                                         - - Met Leu Ser Pro                                                            1                                                                            - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: rHb1.1              beta chain peptide                                                       - - <400> SEQUENCE: 6                                                         - - Met His Leu Ser Pro                                                        1               5                                                            - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide             from horse, donkey, kulan, zebra - #or gundi                             - - <400> SEQUENCE: 7                                                         - - Val Leu Ser Ala Ala Asp Lys Thr Asn Val Ly - #s Ala Ala Trp                1               5 - #                 10                                     - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 45                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     Oligonucleotide- C-term of x gene, - #Xba I site                         - - <400> SEQUENCE: 8                                                         - - cgggaatacg gtctagatca ttaacggtat ttcgaagtca gaacg   - #                      - #45                                                                      - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 95                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     Oligonucleotide- tac promoter, Bam H - #I-Eag I sites                    - - <400> SEQUENCE: 9                                                         - - gatccgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg tg -             #acggataa     60                                                                 - - caatttcaca caggaaatta attaatgctg tctcc       - #                       - #       95                                                                     - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 96                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                    Oligonucleotide- tac promoter, Bam H - #I-Eag I sites                    - - <400> SEQUENCE: 10                                                        - - ggccggagac agcattaatt aatttcctgt gtgaaattgt tatccgctca ca -             #attccaca     60                                                                 - - cattatacga gccgatgatt aattgtcaac agctcg      - #                       - #       96                                                                     - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 64                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                    Oligonucleotide- 5' end of alpha - #with Eco RI, Bam                          HI and Eag I sites                                                       - - <400> SEQUENCE: 11                                                        - - tcggattcga attccaagct gttggatcct tagattgaac tgtctccggc cg -             #ataaaacc     60                                                                 - - accg                 - #                  - #                  - #                 64                                                                  - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 55                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     Oligonucleotide-5' end of beta wi - #th Xba I site                       - - <400> SEQUENCE: 12                                                        - - cggaagccca atctagagga aataatatat gcacctgact ccggaagaaa aa - #tcc              55                                                                        - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 44                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     Oligonucleotide- 3' end of the - #Beta gene with Hind                         III site                                                                 - - <400> SEQUENCE: 13                                                        - - cccgaaacca agcttcatta gtgagctagc gcgttagcaa cacc   - #                      - # 44                                                                      - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 37                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     mutagenesis reverse primer                                               - - <400> SEQUENCE: 14                                                        - - tttaagcttc attagtggta tttgtgagct agcgcgt      - #                       - #      37                                                                      - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 37                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                    mutagenesis reverse primer                                               - - <400> SEQUENCE: 15                                                        - - cagcattaat taacctcctt agtgaaattg ttatccg      - #                       - #      37                                                                      - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 45                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                    mutagenesis reverse primer                                               - - <400> SEQUENCE: 16                                                        - - ggtgcatata tttacctcct tatctagatc attaacggta tttcg   - #                      - #45                                                                      - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 10                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Pme I               linker                                                                   - - <400> SEQUENCE: 17                                                        - - ggtttaaacc                - #                  - #                      - #        10                                                                   - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 58                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     Oligonucleotide upstream of lacI gen - #e                                - - <400> SEQUENCE: 18                                                        - - ggcgaataaa agcttgcggc cgcgttgaca ccatcgaatg gcgcaaaacc tt - #tcgcgg           58                                                                        - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 69                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: downstrea           side of lacI gene                                                        - - <400> SEQUENCE: 19                                                        - - gggcaaatag gatccaaaaa aaagcccgct cattaggcgg gctttatcac tg -             #cccgcttt     60                                                                 - - ccagtcggg                - #                  - #                      - #         69                                                                  - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 54                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer       for                                                                                   ori pBR322 positions 3170-3148                                           - - <400> SEQUENCE: 20                                                        - - ccccgaaaag gatccaagta gccggcggcc gcgttccact gagcgtcaga cc - #cc               54                                                                       - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 42                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer       for                                                                                   pB32 ori positions 2380-2404                                             - - <400> SEQUENCE: 21                                                        - - ggcggtcctg tttaaacgct gcgctcggtc gttcggctgc gg    - #                      - #  42                                                                     - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 28                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: diapha              gene fragment                                                            - - <400> SEQUENCE: 22                                                        - - caatttcaca ggaaattaat taatgctg         - #                  - #                 28                                                                      - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 30                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: diapha              gene fragment                                                            - - <400> SEQUENCE: 23                                                        - - caatttcact aaggaggtta attaatgctg         - #                  - #               30                                                                      - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: beta         gene                                                                                  fragment                                                                 - - <400> SEQUENCE: 24                                                        - - taaagatcta gaggaaataa tatatgcac         - #                  - #                29                                                                     - -  - - <210> SEQ ID NO 25                                                  <211> LENGTH: 33                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: beta         gene                                                                                  fragment                                                                 - - <400> SEQUENCE: 25                                                        - - taatgatcta gataaggagg taaatatatg cac       - #                  -      #         33                                                                     - -  - - <210> SEQ ID NO 26                                                  <211> LENGTH: 16                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: beta               terminus                                                                 - - <400> SEQUENCE: 26                                                        - - ctcgctcact aatgaa             - #                  - #                      - #    16                                                                   - -  - - <210> SEQ ID NO 27                                                  <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: modified            beta terminus                                                            - - <400> SEQUENCE: 27                                                        - - ctcgctcaca aataccacta atgaa          - #                  - #                   25                                                                      - -  - - <210> SEQ ID NO 28                                                  <211> LENGTH: 33                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     oligonucleotides for rHb1.1 mutants                                      - - <400> SEQUENCE: 28                                                        - - ggaggttaat taatgctgtc tgccgccgat aaa       - #                  - #             33                                                                      - -  - - <210> SEQ ID NO 29                                                  <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide        - - <400> SEQUENCE: 29                                                        - - Met Leu Ser Ala Ala Asp Lys                                                1               5                                                            - -  - - <210> SEQ ID NO 30                                                  <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide        - - <400> SEQUENCE: 30                                                        - - Met Leu Ser Ala                                                            1                                                                            - -  - - <210> SEQ ID NO 31                                                  <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide        - - <400> SEQUENCE: 31                                                        - - Met Leu Ser Ser                                                            1                                                                            - -  - - <210> SEQ ID NO 32                                                  <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     oligonucleotide for rHb1.1 mutants                                       - - <400> SEQUENCE: 32                                                        - - ggaggttaat taatggccct gtctccg          - #                  - #                 27                                                                      - -  - - <210> SEQ ID NO 33                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide        - - <400> SEQUENCE: 33                                                        - - Met Ala Leu Ser Pro                                                        1               5                                                            - -  - - <210> SEQ ID NO 34                                                  <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     Oligonucleotide for rHb1.1 mutants                                       - - <400> SEQUENCE: 34                                                        - - ggaggttaat taatgtctct gtctccg          - #                  - #                 27                                                                      - -  - - <210> SEQ ID NO 35                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide        - - <400> SEQUENCE: 35                                                        - - Met Ser Leu Ser Pro                                                        1               5                                                            - -  - - <210> SEQ ID NO 36                                                  <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     oligonucleotide for rHb1.1 mutants                                       - - <400> SEQUENCE: 36                                                        - - ggaggttaat taatgaccct gtctccg          - #                  - #                 27                                                                      - -  - - <210> SEQ ID NO 37                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide        - - <400> SEQUENCE: 37                                                        - - Met Thr Leu Ser Pro                                                        1               5                                                            - -  - - <210> SEQ ID NO 38                                                  <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     oligonucleotide for rHb1.1 mutants                                       - - <400> SEQUENCE: 38                                                        - - ggaggttaat taatggttct gtctccg          - #                  - #                 27                                                                      - -  - - <210> SEQ ID NO 39                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide        - - <400> SEQUENCE: 39                                                        - - Met Val Leu Ser Pro                                                        1               5                                                            - -  - - <210> SEQ ID NO 40                                                  <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:                     oligonucleotide for rHb1.1 mutants                                       - - <400> SEQUENCE: 40                                                        - - ggaggttaat taatggttct gtctgcc          - #                  - #                 27                                                                      - -  - - <210> SEQ ID NO 41                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide        - - <400> SEQUENCE: 41                                                        - - Met Val Leu Ser Ala                                                        1               5                                                            - -  - - <210> SEQ ID NO 42                                                  <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: peptide        - - <400> SEQUENCE: 42                                                        - - Met Leu Ser Ser                                                         __________________________________________________________________________

What is claimed is:
 1. A method for decreasing methylation of anN-terminus of a protein having a proline at amino acid position 4recognized by a methylation enzyme that directs methylation of saidN-terminus comprising:altering said amino acid position 4 to anon-proline residue wherein said protein is at least partiallymethylated at the N-terminus prior to said alteration and has decreasedmethylation after said alteration when said protein is expressed in abacterium.
 2. A method according to claim 1 wherein said protein isselected from the group consisting of hemoglobin, L16 and CheZ.
 3. Amethod according to claim 2 wherein said protein is hemoglobin.
 4. Amethod according to claim 1 wherein said alteration is selected from thegroup consisting of (1) shifting said proline at amino acid position 4by addition or deletion of one or more amino acids upstream of position4 and (2) substitution of the proline at amino acid position
 4. 5. Amethod according to claim 4 wherein said alteration is made by sitedirected mutagenesis of a nucleic acid sequence coding for said protein.6. A method according to claim 4 wherein said alteration is substitutionof the proline at amino acid position 4 with an amino acid selected fromthe group consisting of alanine and serine.
 7. A method according toclaim 4 wherein said addition of one or more amino acids upstream ofamino acid position 4 is adjacent to amino acid position
 2. 8. A methodaccording to claim 7 wherein said additional amino acid is selected fromthe group consisting of alanine, serine, threonine and valine.
 9. Amethod for decreasing methylation of an N-terminal methionine ofrecombinant hemoglobin having a proline at amino acid position 4comprising:altering amino acid position 4 to a non-proline residue, saidalteration being selected from the group consisting of(a) substitutionof the proline at position 4 with alanine, (b) substitution of theproline at position 4 with serine, (c) addition of alanine adjacent toand upstream of amino acid position 2, (d) addition of serine adjacentto and upstream of amino acid position 2, (e) addition of threonineadjacent to and upstream of amino acid position 2, and (f) addition ofvaline adjacent to and upstream of amino acid position 2, wherein saidhemoglobin is at least 20 percent methylated at the N-terminus prior tosaid alteration when said protein is expressed in E. coli.
 10. A methodaccording to claim 1 wherein said N-terminus is an N-terminalmethionine.
 11. A method according to claim 7 wherein said addition ofone or more amino acids adjacent to position 2 is upstream of saidposition 2.